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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 13 November 1998 (signature of the protocol) to 8 March 1999 (date of the study report)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
version of 1984
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
To study the exposure of the water fleas to lithium cryolite, samples of the test solutions were taken and the fluoride content of the samples was measured by cuvette test LCK 323 (Dr. Lange, Tiel, The Netherlands).
On day 0, samples of 10 mL test solution were taken from all bulk solutions and analysed. On day 2 samples of 10 mL were taken from all glass beakers. The samples with nominal concentrations of 2; 4.5; 10; 22 and 50 mg/L were diluted 2; 5; 10; 20 and 25 times respectively with ultrapure water before analysis.
Vehicle:
no
Details on test solutions:
Bulk solutions were prepared on the day 0 before test initiation by dissolving 2.02; 4.47; 10.01; 22.08 and 49.95 mg lithium cryolite in 1000 mL ISO-water to produce solutions with concentrations of about 2; 4.5; 10; 22 and 50 mg/L. The solutions were stirred for 24 hours at 20°C in order to be sure that dissolution was complete. On the day of test initiation the bulk solutions were filtered with filter paper (S&S, 595 1/2).
The test solutions were clear and particles of the test substance were not visible.
Test organisms (species):
Daphnia magna
Details on test organisms:
Water fleas (Daphnia magna, Crustacea) used in this test originated from the Daphtoxkit F. magna. the culture is in house since January 1998 and originates from Bio International, The Netherlands. Water fleas of the same age are cultured in vessels with 2 Litre Elendt M4 medium. They are fed with an algae suspension of chlorella vulgaris.
The water fleas are cultured in a climate chamber with a temperature set point of 20°C. The light regime is 16 hours light and 8 hours darkness per day. The light is switched on at 6 a.m. Each week new culture of water fleas is started and water fleas older than 3 weeks are removed. The age of the water fleas used in the test was 0-24 hours at the start of the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
no data
Test temperature:
The air temperature in the climate chamber was recorded continuously during the experiment and this measured air temperature ranged between 20 and 21°C.
The measured temperature in the test solutions at test initiation and at test termination ranged between 19.6°C and 20.9°C.
pH:
At test initiation the pH of the bulk solutions was measured and was 7.9; 7.9; 7.8; 7.7; 7.1 and 7.1 respectively and the pH was therefore not corrected.
The measured pH in the test solutions at test initiation and at test termination ranged between 7.1 and 7.9.
Dissolved oxygen:
The measured oxygen content of the test solutions at test initiation and at test termination ranged from 8.4 to 8.9 mg/L, and therefore was higher than the minimum value of 5.6 mg/L.
Salinity:
No data
Nominal and measured concentrations:
Based on the results of the preliminary studies, the final study was conducted with nominal test concentrations of 0; 2; 4.5; 10; 22 and 50 mg lithium cryolite per litre ISO-water and the mean measured concentrations were 2.6; 5.4; 11.5; 22.5 and 45.3 mg/L respectively.
The difference between the nominal and the mean measured concentrations was > 20% at one concentration (2 mg/L). As at this concentration no effect was found and the difference in the other concentrations was < 20% the results are based on nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass beakers placed in a climate chamber at 20°C.
- Material, size, headspace, fill volume: 200 mL test solution in each beaker
- Aeration: no data
- Renewal rate of test solution (frequency/flow rate): not applicable (static)
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: 2 mL / each animals
- the water fleas were not fed during the test.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO-water prepared in ultra pure water (i.e. water prepared by reversed osmosis, followed by anion, cation and mixed bed chromatography and filtered through a 0.22 µm cellulose filter; the water is prepared according to Probis no. 24 33 02 and the conductivity of the water is less than 4 µS/cm).
- Total organic carbon: no data
- Culture medium different from test medium: As Elendt medium contains sulfide which can react with the test compound it was decided to perform the test in ISO-water and a reference test with potassium dichromate was conducted to control the sensitivity of water fleas in ISO-water (see results with reference substance)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours dark (light from 6 a.m. to 10 p.m.).
- Light intensity: no data

EFFECT PARAMETERS MEASURED :
The number of mobile and immobile water fleas and the number of water fleas showing sublethal effects was recorded 24 and 48 hours after test initiation. Immobile water fleas are those animals which are not able to swim within 15 seconds after gentle agitation of the test flask.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
18.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: observed at 10; 22 and 50 mg/L (see table 1)
- Observations on body length and weight: not performed
- Other biological observations: no data
- Mortality of control: no immobility observed in the control
- Other adverse effects control: no adverse effect observed in the control
- Abnormal responses: none
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 24h EC50 = 1.23 mg/L (95% CI 1.15 - 1.32 mg/L)
According to ISO 6341 (1996) the 24h EC50 of potassium dichromate should be within the range of 0.6 - 1.7 mg/L.
Reported statistics and error estimates:
The NOEC was determined with Fisher's Exact Test (Sokal et al., 1981).
The 48h EC50 was calculated with probit analysis (Finney, 1980). No 95% confidence interval could be determined.

Deviation from protocol:

According to the protocol, on day 0 the pH, oxygen content and temperature of the ISO-water and the highest concentration would be measured. On day 0 the pH, oxygen content and temperature were measured in one glass beaker per test concentration. To the opinion of the study director this deviation did not influence the integrity of the study.

Table 1: Number of mobile water fleas during the test

Nominal concentration

(mg/L)

Number of mobile water fleas (1)

Mean immobility at test termination

(%)

0 hours

24 hours

48 hours

0

10

10

10

10

10

10

10

10

10

0

 

2

10

10

10

10

10

10

10

10

10

0

 

4.5

11

10

10

11

10

10

11

10

10

0

10

10

10

10

10 (1)

10

10(2)

10 (1)

10

10

0

22

10

10

10

6

6

8

1

2

1

87

50

10

10

10

4 (2)

1 (1)

3

2 (2)

0

0

93

(1)   Between brackets the number of mobile water fleas showing sublethal effects is given.

Table 2: Measured concentration of Lithium cryolite in test solutions

Nominal concentration

(mg/L)

Dilution factor

Day 0

Day 2

Measured fluoride concentration

(mg/L)

Calculated lithium cryolite concentration (1)

(mg/L)

Measured fluoride concentration

(mg/L)

Calculated lithium cryolite concentration (1)

(mg/L)

0

1

0

n.d.

0

0

0

n.d.

n.d.

n.d.

2

2

0.888

2.7

0.795

0.827

0.824

2.4

2.5

2.5

4.5

5

0.699

5.3

0.761

0.709

0.689

5.8

5.4

5.2

10

10

0.793

12

0.734

0.739

0.704

11.1

11.2

10.6

22

20

0.757

22.9

0.724

0.752

0.709

21.9

22.8

21.4

50

25

1.2

45.8

1.19

1.12

1.21

45.4

42.7

46.2

(1)   Lithium cryolite concentration corrected for the dilution factor

n.d. = not detected.

 
Validity criteria fulfilled:
yes
Remarks:
The control mortality was less than 10% during the study and the dissolved oxygen concentration in the test solutions was higher than 60% of the air saturation value.
Conclusions:
The 48hEC50 of Lithium cryolite to Daphnia magna was found to be 18.2 mg/L.
Executive summary:

The acute toxicity of lithium cryolite to water fleas (Daphnia magna) was tested following the OECD testing guideline 202 (1984) and according to the GLP.

Based on a preliminary range-finding study, the test was conducted at nominal concentrations of 0; 2; 4.5; 10; 22 and 50 mg/L. Three glass beakers, containing each 200 mL test solution, were prepared per concentration and control. At test initiation 10 water fleas were added to each glass beakers and the glass beakers were placed in a climate chamber at 20°C. The experiment was terminated after 48 hours of exposure.

To study the exposure of the water fleas to the test compound, samples of the test solutions were taken on days 0 and 2 and the fluoride content of the samples was measured by cuvette test LCK 323. The difference between the nominal and the mean measured concentrations was > 20% at one concentration (i.e. 2 mg/L). As at this concentration no effect was found and the difference in the other concentrations was < 20% the results are based on nominal concentrations.

No immobility was observed in the control glass beakers. The dissolved oxygen concentration in the test solutions was higher than 60% of the air saturation value. Therefore, the OECD validity criteria were fulfilled.

The 48h NOEC based on immobility was determined with Fisher’s Exact Test and was equal to 10 mg/L.

The 48h EC50 based on immobility was calculated with probit analysis and was equal to 18.2 mg/L. No 95% confidence interval could be determined.

 

Description of key information

A reliable acute toxicity test is available, performed under static conditions.

Based on the immobility observed, the 48h EC50 was determined to be equal to 18.2 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
18.2 mg/L

Additional information

One reliable key study is available for this endpoint. In this study, the acute toxicity of lithium cryolite to water fleas (Daphnia magna) was tested following the OECD testing guideline 202 (1984) and according to the GLP.

Based on a preliminary range-finding study, the test was conducted at nominal concentrations of 0; 2; 4.5; 10; 22 and 50 mg/L. Three glass beakers, containing each 200 mL test solution, were prepared per concentration and control. At test initiation 10 water fleas were added to each glass beakers and the glass beakers were placed in a climate chamber at 20°C. The experiment was terminated after 48 hours of exposure.

To study the exposure of the water fleas to the test compound, samples of the test solutions were taken on days 0 and 2 and the fluoride content of the samples was measured by cuvette test LCK 323. The difference between the nominal and the mean measured concentrations was > 20% at one concentration (i.e. 2 mg/L). As at this concentration no effect was found and the difference in the other concentrations was < 20% the results are based on nominal concentrations.

No immobility was observed in the control glass beakers. The dissolved oxygen concentration in the test solutions was higher than 60% of the air saturation value. Therefore, the OECD validity criteria were fulfilled.

The 48h NOEC based on immobility was determined with Fisher’s Exact Test and was equal to 10 mg/L.

The 48h EC50 based on immobility was calculated with probit analysis and was equal to 18.2 mg/L. No 95% confidence interval could be determined.